Vol. 28, No. 3

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1990, p. 504-507

0095-1137/90/030504-04$02.00/0 Copyright © 1990, American Society for Microbiology

Rotavirus Serotypes 6 and 10 Predominate in Cattle D. R.

SNODGRASS,1* T. FITZGERALD,' I. CAMPBELL,' F. M. M. SCOTT,' G. F. BROWNING,' D. L. MILLER,' A. J. HERRING,' AND H. B. GREENBERG2 Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JH, Scotland,' and Stanford University School of Medicine, Stanford, California 9430551002

Received 8 August 1989/Accepted 21 November 1989

Calf fecal rotavirus strains were serotyped in enzyme-linked immunosorbent assays, using monoclonal antibodies to the VP7s of serotypes 1, 2, 3, 5, and 6 and to the VP4 of B223 (designated serotype 10). Sixty-six percent of 162 samples were typed as serotype 6, and 7% were serotyped as serotype 10. Most of the untyped strains did not react with a monoclonal antibody directed to a common VP7 epitope, indicating insufficient virus present in the samples. However, seven untyped samples that did react with this antibody were adapted to culture and typed, and six of these also proved to belong to serotype 6 or 10. Two of these viruses belonged to a monotype within serotype 6 that did not react with the serotype 6 monoclonal antibody. The seventh isolate reacted in cross-neutralization tests with serotype 8 viruses. Bovine rotaviruses from the United Kingdom, Federal Republic of Germany, and Japan that had been shown previously to be distinct from serotype 6 were compared in neutralization tests with B223 from the United States. These viruses proved to be a closely reacting group distinct from all other rotavirus serotypes, justifying the establishment of serotype 10 as the second major type of bovine rotavirus.

6), human 69M (serotype 8), and human WI-61 (serotype 9) (6, 13, 15). Bovine rotaviruses used other than serotype 6 strains were strain 678 isolated in our laboratory (19), strain B223 isolated in the United States (potentially serotype 10 [29]), strain KK3 isolated in Japan (17), and V1005 isolated in the Federal Republic of Germany (1). In addition, strain 411 had been isolated in our laboratory and, although the isolate itself had been discarded due to subsequent contamination with another rotaviral strain, antiserum was available (24). A total of 162 samples of calf feces from throughout the United Kingdom was tested. Ail were group A rotaviruses, shown by detection of double-stranded RNA in sodium dodecyl sulfate-polyacrylamide gels, and only samples containing an electrophoretype of 11 segments were included (12). Hyperimmune antisera were prepared in rabbits from a rotavirus-free colony by conventional techniques. Serotyping ELISAs. Serotyping ELISAs were similar to those used for serotyping human rotaviruses (8, 26). Separate Nunc ELISA microplates were coated with 1/5,000 dilution of mouse ascitic fluid containing each MAb in carbonate-bicarbonate buffer overnight at 4°C; the plates were postcoated with 10% ovalbumin for 1.5 h. The standard rotavirus and test fecal suspensions were added to each plate, and a 1/1,000 dilution of a polyclonal rabbit antiserum to calf rotavirus was added, the same serum being used for all serotypes. Finally, a sheep anti-rabbit immunoglobulin G conjugated with alkaline phosphatase was added before the reaction was developed with appropriate substrate. Phosphate-buffered saline plus 0.05% Tween 20 was used as washing buffer between each stage, and the reagent diluent was phosphate-buffered saline-10% ovalbumin-0.05% Tween 20. Each fecal sample was tested in ELISAs, using VP7 MAbs specific to serotypes 1, 2, 3, 5, and 6, a VP4 MAb to B223, and the common VP7 MAb 60 Ai. MAbs to other serotypes were not available to us. The results were expressed as the mean optical density at 405 nm (OD405) in two wells. These means for each sample

Rotaviruses are the major infectious cause of diarrhea in calves in many countries (2, 9, 20, 25). Neutralizing antibody production is mediated by epitopes on both outer capsid proteins VP4 (coded for by gene segment 4) and VP7 (from gene segment 8 or 9), but serotypes are largely defined on the dominant VP7 glycoprotein. Nine serotypes have been generally accepted (11, 13), but in addition, B223 bovine virus has been proposed as serotype 10 (I. Holmes, personal communication). Most bovine rotaviruses that have been typed have belonged to serotype 6, but a few distinct viruses have been isolated (1, 2, 17, 24, 27, 29). These have usually been characterized only as distinct from serotype 6, and their potential relationships to each other have not been studied. In spite of their importance in humans and in mammalian species of veterinary interest, effective rotavirus vaccination has been achieved routinely only in cattle, by passive immunization through dam vaccination (16, 23). This has been accomplished in spite of the scanty knowledge of serotype prevalence and is perhaps due to a strong heterotypic immune response (4, 5, 24). For epidemiological and immunological reasons, it is therefore important to define further naturally occurring bovine rotavirus serotypes. The production of monoclonal antibodies (MAbs) to the serotype-specific epitopes of the VP7s of a range of rotaviruses has allowed the development of enzyme-linked immunosorbent assays (ELISAs) to serotype rotaviruses in fecal samples (8, 22, 26). This technique allowed the further characterization of bovine rotaviruses in this study. young

MATERIALS AND METHODS Rotavirus strains and antisera. Reference rotavirus serotype strains used were human Wa (serotype 1), human DS-1 (DS-1 x UK reassortant, containing VP7 of DS-1, serotype 2), rhesus rotavirus (serotype 3), human ST-3 (serotype 4), porcine OSU (serotype 5), bovine UK and NCDV (serotype *

Corresponding author. 504

ROTAVIRUS SEROTYPES 6, 8, AND 10 IN CATTLE

VOL. 28, 1990 TABLE 1. Serotyping ELISA results with cell culture rotaviruses of known serotype OD40_5 (10-2) in ELISA to MAb' UK7 4F8 5B8

Virus (serotype)

2C9

IC10

Wa(1) DS-1(2) RRV (3) OSU (5) UK (6) B223 (10)

56 6 40 15 15 14

2 82 4 2 4 4

3 3 98 3 4 4

1 1 2 57 2 2

5 4 5 3 74 3

505

TABLE 2. Results of typing 162 bovine rotaviruses from fecal samples B223/3

1 5 il 7 4 191

a Homologous values are shown in boldface type.

for each serotype were divided by the lowest serotyping OD405 obtained with that sample: values of >5, and with a test OD405 of >0.10 and no heterologous reactions, were considered to be serotyped. Culture and serology. Cell culture-adapted strains of rotavirus were cultivated in MA104 cells as described previously (19), and selected strains were adapted to growth in MA104 cells by standard techniques. Neutralization tests were microdilution fluorescent focus reduction tests, with endpoint titer determined as 60% reduction in foci. MAbs. Neutralizing MAbs 2C9, IC10, 4F8, and 5B8 to VP7 of rotavirus serotypes 1, 2, 3, and 5, respectively, and MAb 60 Ai to a common non-neutralizing VP7 epitope were used (21, 22; H. B. Greenberg, unpublished data). MAb UK/7 to strain UK was prepared by standard techniques, using spleen from immunized BALB/c mice fused to NS-0 cells. The hybridoma cultures were screened by neutralization test, and selected hybridomas were terminally diluted three times before amplification in the peritoneal cavities of Pristane-treated seronegative BALB/c mice. The MAb was determined to be of immunoglobulin M isotype by monoclonal isotyping kit (Amersham Corp.). The neutralization titer of UK/7 of both UK and NCDV viruses was >105. Since these two serotype -6 viruses share common VP7s but not VP4s (14, 18), UK/7 was considered to be VP7 specific. MAb B223/3 was produced by similar techniques and was of immunoglobulin G2b isotype. To determine protein specificity, a UK x B223 reassortant (B-UK-2) was isolated. This reassortant contained the VP4 gene from UK and the VP7 gene from B223. The neutralization titers of MAb B223/3 to viruses UK, B223, and B-UK-2 were

Rotavirus serotypes 6 and 10 predominate in cattle.

Calf fecal rotavirus strains were serotyped in enzyme-linked immunosorbent assays, using monoclonal antibodies to the VP7s of serotypes 1, 2, 3, 5, an...
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